DESCRIPTION: (Applicant's Abstract) It has been estimated that 40 percent of the brain synapses are GABAergic. However, little is known about the mechanisms by which GABAA receptors (GABAAR) are concentrated at inhibitory GABAergic Gray's type 2 synapses. The long-term objective of this proposal is to reveal the molecular components of the GABAergic Gray's type 2 postsynaptic density and to understand the molecular mechanisms involved in clustering and anchoring GABAAR at the postsynaptic membrane. The specific aims of this proposal are directed towards revealing the cytoplasmic proteins that directly bind to GABAAR and to test whether these proteins are responsible for the clustering and/or localization of the GABAAR at synapses and/or other membrane domains. The aims will test the hypotheses that 1) a novel GABAAR protein is involved in GABAAR clustering and/or anchoring at GABAergic synapses; 2) in neurons, the interaction of the beta3 GABAAR subunit with a guanine nucleotide exchange protein is necessary for GABAAR transport to the cell surface; and 3) the novel proteins that interact with various GABAAR subunits in the yeast two-hybrid screens also interact with the GABAAR in neurons and are involved in receptor clustering or anchoring at synapses. Yeast two-hybrid assays are being used in our laboratory to identify GABAAR binding proteins. The functionality of the interaction between GABAAR and binding proteins will be tested by various in vitro biochemical assays as well as by in vivo (in culture) assays including primary hippocampal neuronal cultures and cotransfected host cells. The synaptic colocalization of the GABAA receptors and interacting proteins will be studied by both light microscopic immunofluorescence and electron microscopic immunogold immunocytochemistry using specific antibodies to GABAAR subunits and interacting protein. The GABAAR and interacting protein amino acid sequences involved in protein-protein interactions will be identified. These studies will help to better understand the molecular components and interactions involved in the structure, function and modulation of the inhibitory GABAergic synaptic transmission as well as the mechanisms involved in neuronal connectivity and synaptic plasticity. It is predicted that the inappropriate synaptic and extrasynaptic localization of the GABAAR, affects GABAergic synaptic function and brain development, leading to neurological and mental disorders.